Variants of humanized immunomodulatory monoclonal antibodies

ABSTRACT

The present invention relates to humanized monoclonal antibodies, pharmaceutical compositions that include the same, and use thereof for the treatment of a variety of indications, particularly cancer and immunodeficiency disorders. In particular, the present invention provides modified antibodies or fragments thereof having specific amino acid modifications compared to the humanized monoclonal immunomodulatory antibody termed hBAT-1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/556,018filed Jul. 23, 2012, which claims the benefit of U.S. application No.61/511,055 filed Jul. 24, 2011.

FIELD OF THE INVENTION

The present invention relates to the field of immunotherapy and morespecifically concerns humanized monoclonal antibodies useful for therapyof a variety of indications, particularly in the treatment of cancer andimmunodeficiency disorders.

BACKGROUND OF THE INVENTION

The rapid increase of knowledge in recent years about the molecular andcellular bases of immune regulation, particularly at the level of T cellresponses, provides a new arsenal of immunotherapeutic approachesincluding the development of anti-tumor vaccines. Certain monoclonalantibodies were shown to have immunomodulatory activity including theability to bind determinants on the surface of T cells and to induceproliferation, activation, maturation or differentiation of these cells.

BAT (also referred to as mBAT-1 or BAT-1) is a murine monoclonalantibody generated against a membrane preparation of a Burkitt lymphomacell line (Daudi) that was shown to exhibit antitumor andimmunostimulatory effects towards various types of tumors (Hardy et al.,2001, Int. J. Oncol. 19:897). This monoclonal antibody was initiallydisclosed in U.S. Pat. No. 5,897,862 to Hardy et al. BAT-1 is secretedby the hybridoma cell line having CNCM Accession No. I-1397. Theimmunomodulatory effect of murine BAT was studied also in vitro. MurineBAT activates CD4+ T cells and induces the secretion of IFN-γ from thesecells (Hardy et al., 2000, Int. Immunol. 12:1623 and Quaglino E. et al.,2005, Vaccine 9:23(25):3280-7). In addition, it was found that BATtriggers the proliferation of T cells and increases their cytolyticactivity (Hardy, B. et al., 1997, Hum. Antibodies, 8:95).

The polynucleotide and amino-acid sequences of murine BAT are disclosedin WO 00/58363, to Hardy et al., and U.S. Pat. No. 7,695,715.

A number of humanized monoclonal antibodies (mAbs) based on murine BATare disclosed in U.S. Pat. No. 7,332,582, the contents of which areincorporated herein by reference. In particular embodiments of U.S. Pat.No. 7,332,582, the humanized monoclonal antibodies comprise a lightchain variable region selected from SEQ ID NO: 1-4 (denoted BATRK_(D),BATRK_(A), BATRK_(B) and BATRK_(C), respectively) and a heavy chainvariable region selected from SEQ ID NO: 5-9 (denoted BATRH_(C),BATRH_(A), BATRH_(B), BATRH_(D) and BATRH_(E), respectively). The aminoacid sequence of the light and heavy chain antibody variants aredepicted in Table 1 herein below. The residues which differ in saidvariable regions are depicted with a gray background (positions 2, 30,69, 77, 97 and 98 of the light region and positions 35, 69, 70 and 71 ofthe heavy chain).

According to U.S. Pat. No. 7,332,582, the humanized monoclonal BATantibodies appear to induce a greater antitumor effect than thoseinduced by the parent murine BAT antibody. Among various model systemstested, the BAT antitumor activity was studied in SCID (severe combinedimmunodeficiency disease) mice, beige mice that are deficient in NKcells and nude mice that are deficient in T cells (Hardy, B., 1997,Proc. Natl. Acad. Sci. USA 94:5756). All mice were injectedintravenously with murine B16 melanoma cells that subsequently developedtumors in the lungs. BAT antibodies exerted an antitumor effect only inSCID mice that were engrafted with either murine or human lymphocytes.In the athymic nude mice and the beige mice BAT antibodies exerted anantitumor activity, though this activity was less effective as comparedto the antitumor activity of BAT antibodies in the wild- type mice.

It should be borne in mind that BAT antibodies are not expected totarget the tumor cells themselves but rather the immune-functioningcells of the subject or patient, in order to modulate the immuneresponse in a beneficial way.

Berger et al. (2008) discloses administration of the humanizedmonoclonal antibody CT-011, which is based on mBAT-1, to patients withadvanced hematologic malignancies, and associated pharmacokinetics(Berger et al. Clin. Cancer Res. 2008;14(10) May 15, 2008).

WO 09/101611 relates to methods for inhibiting tumor growth, increasingsurvival of a subject having a tumor and inducing protection againsttumor recurrence in a mammal, comprising administering a humanizedmonoclonal antibody comprising CDR regions derived from the murinemonoclonal antibody designated mBAT-1, in combination with at least onechemotherapeutic agent.

Nowhere in the background art is it taught or suggested that use of ahumanized mBAT-1 monoclonal antibody comprising at least one sitespecific amino acid modification will be advantageous for the therapy ofa variety of indications, particularly in the treatment of cancer andimmuno-deficiency related diseases and disorders.

SUMMARY OF THE INVENTION

The present invention provides mutated humanized monoclonal antibodiesor fragments thereof, having site specific amino acid modificationsincluding but not limited to, amino acid substitutions and deletions,compared to the known humanized monoclonal antibodies hBAT-1. Theinvention further provides pharmaceutical compositions comprising themodified antibodies or fragments thereof, and use thereof for thetreatment of cancer and immunodeficiency disorders. In some embodiments,the modified antibodies of the invention retain the immunomodulatoryactivity of hBAT-1, bind B lymphoblastoid cells and induce proliferationand activation of peripheral blood lymphocytes.

The antibodies of the present invention demonstrate superior features,e.g., enhanced bioactivity and stability and/or reduced immunogenicity,by virtue of a specific modification of at least one amino acid of thehBAT-1 antibody variants, as detailed herein below.

Table 1 lists the amino acid sequences of the variable and constantregions of the light and heavy chain of hBAT-1 antibody variants. The atleast one amino acid which, in some embodiments, is substituted toproduce the antibodies of the invention, is depicted in bold. Inparticular embodiments, the at least one amino acid is selected from thegroup consisting of: the amino acids in positions 5, 20, 71, 75, 76, 93and 97 of SEQ ID NO: 1; the amino acids in positions 54, 55 and 107 ofSEQ ID NO: 5, amino acids in positions 157, 158, 169, 170 of SEQ ID NO:11; and the amino acids in positions 120, 124, 159, 160, 203, 204, 221,222, 225, 252, 270, 271, 280, 281, 297, 298, 315, 316, 384, 385, 399,400, 401, 402, 434, 435, 447 and 428 of SEQ ID NO: 13.

TABLE 1 The variable and constant region of the known hBAT-1 light and heavy chain Ig SEQ ChainRegion Amino acid sequence ID NO: Light Chain Variable

BATRK_(D)  1 Variable

BATRK_(A)  2 Variable

BATRK_(B)  3 Variable

BATRK_(C)  4 Constant RTVAAPSVFIFPPSDEQLKSGTASVVCLLN 10NFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Variable (BATRK_(D)) + Constant

11 Heavy Chain Variable

BATRH_(C)  5 Variable

BATRH_(A)  6 Variable

BATRH_(B)  7 Variable

BATRH_(D)  8 Variable

BATRH_(E)  9 Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVK 12DYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Variable(BATRH_(C)) + Constant

13

According to one aspect, the present invention provides an antibody orantigen binding fragment thereof comprising a light chain variableregion selected from the group consisting of SEQ ID NO: 1, 2, 3, and 4,and a heavy chain variable region selected from the group consisting ofSEQ ID NO: 5, 6, 7, 8, and 9, wherein the antibody or antigen bindingfragment thereof comprises at least one amino acid substitution at aposition selected from: Phe 97 and Phe 93 of the light chain variableregion to Ala, Leu or Val, or a substitution of Trp 107 of the heavychain variable region to Ala, Leu, Val or Tyr.

In some embodiments of the invention, the antibody or antigen bindingfragment thereof comprises an amino acid substitution of Phe 97 of thelight chain variable region and an amino acid substitution of Tip 107 ofthe heavy chain variable region. In another embodiment, the antibody orantigen binding fragment thereof comprises an amino acid substitution ofPhe 93 of the light chain variable region and an amino acid substitutionof Trp 107 of the heavy chain variable region.

In another embodiment, the light chain variable region comprises theamino acid sequence selected from the group consisting of: SEQ ID NO:14, 15, 16, 17, 18, 19, 20 and 21. In one embodiment, the light chainvariable region comprises the amino acid sequence as set forth in SEQ IDNO: 14. In another embodiment, the light chain variable region comprisesthe amino acid sequence as set forth in SEQ ID NO: 15. In a particularembodiment, the light chain variable region comprises the amino acidsequence as set forth in SEQ ID NO: 29. In yet another particularembodiment, the light chain variable region comprises the amino acidsequence as set forth in SEQ ID NO: 30.

In another embodiment, the heavy chain variable region comprises theamino acid sequence selected from the group consisting of: SEQ ID NO:24, 25, 26, 27 and 28. In a particular embodiment, the heavy chainvariable region comprises the amino acid sequence as set forth in SEQ IDNO: 24. In yet another particular embodiment, the heavy chain variableregion comprises the amino acid sequence as set forth in SEQ ID NO: 31.

In yet another embodiment, the antibody or antigen binding fragmentthereof further comprises at least one amino acid substitution at aposition selected from the group consisting of: Thr 5, Thr 20, Cys 71,Asn 75, Ser 76 of the light chain variable region, and Asp 54 and Ser 55of the heavy chain variable region.

According to another aspect, the present invention provides an antibodyor antigen binding fragment thereof comprising a light chain variableregion selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4,and a heavy chain variable region selected from the group consisting ofSEQ ID NO: 5, 6, 7, 8 and 9, wherein the antibody or antigen bindingfragment thereof comprises at least one amino acid modification at aposition selected from the group consisting of: Thr 5, Thr 20, Cys 71,Asn 75, Ser 76, Phe 93 and Phe 97 of the light chain variable region andAsp 54, Ser 55 and Trp 107 of the heavy chain variable region. Eachpossibility represents a separate embodiment of the present invention.

According to some embodiments of the invention, the amino acidmodification is an amino acid substitution. According to one embodiment,the amino acid substitution is a conservative substitution. According toanother embodiment, the amino acid substitution is a non-conservativesubstitution. According to another embodiment, the amino acidmodification is an amino acid deletion. According to some embodiments,the modified antibodies of the invention comprise a combination of aminoacid modifications, such as, an amino acid substitution of one residueand an amino acid deletion of another residue. According to anotherembodiment, the modified antibody comprises at least one amino acidsubstitution and at least one amino acid deletion.

According to various embodiments of the invention, the modified antibodyor fragment thereof comprises a combination of a heavy chain variableregion and a light chain variable region, the combination is selectedfrom the group consisting of: SEQ ID NO: 5/SEQ ID NO: 1; SEQ ID NO:6/SEQ ID NO: 2; SEQ ID NO: 7/SEQ ID NO: 2; SEQ ID NO: 7/SEQ ID NO: 3;SEQ ID NO: 8/SEQ ID NO: 3; and SEQ ID NO: 7/SEQ ID NO: 1. According toparticular embodiments of the invention, the antibody or antigen bindingfragment thereof comprises a combination of variable regionscorresponding to SEQ ID NO: 5/SEQ ID NO: 1. The combination of the heavyand light chain variable region comprises, in one embodiment, at leastone amino acid modification at a position selected from the groupconsisting of: Thr 5, Thr 20, Cys 71, Asn 75, Ser 76, Phe 93 and Phe 97of the light chain variable region and Asp 54, Ser 55 and Trp 107 of theheavy chain variable region. In another embodiment, the combination ofthe heavy and light chain variable region comprises at least one aminoacid modification at a position selected from: Phe 93 and Phe 97 of thelight chain variable region and Trp 107 of the heavy chain variableregion. Each possibility represents a separate embodiment of the presentinvention.

According to another embodiment of the invention, the antibody comprisesa light chain constant region as set forth in SEQ ID NO: 10. Accordingto yet another embodiment, the antibody comprises a modified lightconstant region comprising at least one amino acid modification at aposition selected from the group consisting of: Asn 51, Ser 52, Asp 63and Ser 64 of SEQ ID NO: 10. Each possibility represents a separateembodiment of the present invention.

According to another embodiment of the invention, the antibody comprisesa light chain as set forth in SEQ ID NO: 11. According to yet anotherembodiment, the antibody comprises a modified light chain as set forthin SEQ ID NO: 11 comprising an amino acid modification at a positionselected from the group consisting of: Asn 157, Ser 158, Asp 169 and Ser170 of SEQ ID NO: 11. Each possibility represents a separate embodimentof the present invention.

According to another embodiment of the invention, the antibody comprisesa heavy chain constant region as set forth in SEQ ID NO: 12. Accordingto yet another embodiment, the antibody comprises a modified heavy chainconstant region comprising at least one amino acid modification at aposition selected from the group consisting of: Thr 3, Ser 7, Asn 42,Ser 43, Asn 86, His 87, Asp 104, Lys 105, Thr 108, Met 135, Asp 153, Pro154, Asp 163, Gly 164, Asn 180, Ser 181, Asn 198, Gly 199, Asn 267, Gly268, Asp 282, Ser 283, Asp 284, Ser 285, Asn 317, His 318, Lys 330 andMet 311 of SEQ ID NO: 12. Each possibility represents a separateembodiment of the present invention.

According to another embodiment of the invention, the antibody comprisesa heavy chain as set forth in SEQ ID NO: 13. According to yet anotherembodiment, the antibody comprises a heavy chain as set forth in SEQ IDNO: 13 comprising at least one amino acid modification at a positionselected from the group consisting of: Thr 120, Ser 124, Asn 159, Ser160, Asn 203, His 204, Asp 221, Lys 222, Thr 225, Met 252, Asp 270, Pro271, Asp 280, Gly 281, Asn 297, Ser 298, Asn 315, Gly 316, Asn 384, Gly385, Asp 399, Ser 400, Asp 401, Ser 402, Asn 434, His 435, Lys 447 andMet 428 of SEQ ID NO: 13. Each possibility represents a separateembodiment of the present invention.

According to another aspect the present invention provides an antibodyor antigen binding fragment thereof, the antibody comprises a lightchain variable region selected from the group consisting of SEQ ID NO:1, 2, 3 and 4 and or a heavy chain variable region selected from thegroup consisting of SEQ ID NO: 5, 6, 7, 8 and 9, or an amino acidsequence at least 85% identical thereto, wherein the antibody comprisesat least one amino acid modification in a position selected from thegroup consisting of:

(i) Thr 5, Thr 20, Cys 71, Asn 75, Ser 76, Phe 93 and Phe 97 of thelight chain variable region having the amino acid sequence of SEQ ID NO:1-4;

(ii) Asp 54, Ser 55 and Trp 107 of the heavy chain variable regionhaving the amino acid sequence of SEQ ID NO: 5-9;

(iii) Asn 51, Ser 52, Asp 63 and Ser 64 of the light chain constantregion having the amino acid sequence of SEQ ID NO: 10; and

(iv) Thr 3, Ser 7, Asn 42, Ser 43, Asn 86, His 87, Asp 104, Lys 105, Thr108, Met 135, Asp 153, Pro 154, Asp 163, Gly 164, Asn 180, Ser 181, Asn198, Gly 199, Asn 267, Gly 268, Asp 282, Ser 283, Asp 284, Ser 285, Asn317, His 318, Lys 330 and Met 311 of the heavy chain constant regionhaving the amino acid sequence of SEQ ID NO: 12.

In another embodiment of the invention, the antibody comprises a lightchain variable region selected from the group consisting of SEQ ID NO:1, 2, 3 and 4, or a heavy chain variable region selected from the groupconsisting of SEQ ID NO: 5, 6, 7, 8 and 9, or an amino acid sequence atleast 88%, at least 90%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or 99% identicalthereto, wherein each possibility represents a separate embodiment ofthe present invention.

According to various embodiments of the invention, the antibody orantigen binding fragment thereof comprises a combination of modifiedamino acids, including but not limited to, at least two amino acidmodifications, at least three amino acid modifications, at least fouramino acid modifications or at least five amino acid modificationswherein each possibility represent a separate embodiment of the presentinvention.

According to another embodiment, the antibody or antigen bindingfragment thereof comprises at least one amino acid modification at aposition selected from the group consisting of: Thr 5, Thr 20, Cys 71,Asn 75, Ser 76, Phe 93 and Phe 97 of SEQ ID NO: 1-4; and at least oneamino acid modification at a position selected from the group consistingof: Asp 54, Ser 55 and Trp 107 of SEQ ID NO: 5-9. Each possibilityrepresents a separate embodiment of the present invention.

According to another embodiment, the light chain variable region has oneamino acid modification selected from the group consisting of: Thr 5,Thr 20, Cys 71, Asn 75, Ser 76, Phe 93 and Phe 97 of any one of SEQ IDNO: 1, 2, 3, 4 or 11. According to yet another embodiment, the heavychain variable region has one modified amino acid selected from thegroup consisting of: Asp 54, Ser 55 and Trp 107 of any one of SEQ ID NO:5, 6, 7, 8, 9 or 13.

In a particular embodiment, the amino acid modification is at positionCys 71 of any one of SEQ ID NO: 1, 4 or 11. In another embodiment, Cys71 is substituted with an amino acid selected from the group consistingof: Ser, Val, Gly, Thr, Ile, Leu and Ala. Without wishing to be bound byany theory or mechanism of action, the substitution of said Cystineresidue reduces the risk of undesirable disulfide bonds.

Without wishing to be bound by any theory or mechanism of action,modifying one or both amino acid of the following amino acid pairs:Asn-Ser; Asp-Ser; Asn-His; or Asn-Gly, reduces the risk of deamidationthereby increasing the solubility of the antibody of the invention. In aspecific embodiment, the amino acid modification is at a positionselected from the group consisting of: Asn 75, Ser 76 of any one of SEQID NO: 1, 2, 3, 4 or 11. In another embodiment, the amino acidmodification is at a position selected from the group consisting of: Asn157, Ser 158 of SEQ ID NO: 10. In another embodiment, the amino acidmodification is at a position selected from the group consisting of: Asn159, Ser 160, Asn 297, Ser 298 of SEQ ID NO: 11.

In one embodiment, the amino acid pair Asn-Ser is a substituted withSer-Asn. In another embodiment, the amino acid pair Asn-Ser issubstituted with any amino acid pair other than Asp-Ser; Asn-His; orAsn-Gly. In particular embodiments, Asn is substituted with any aminoacid selected from Ala, Val, Ser, Leu, Gly, Glu, or Ser. In anotherparticular embodiment, Asn is substituted with any amino acid other thanGln or Asp. In particular embodiments, Ser is substituted with any aminoacid selected from Ala, Val, Leu, Gly, Ile, Thr or Asn. In yet anotherparticular embodiment, Ser is substituted with any amino acid selectedfrom Ala, Val, Leu, Gly, Ile or Asn. In another particular embodiment,Ser is substituted with any amino acid other than Gly. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the amino acid modification is at a positionselected from the group consisting of: Asp 54, Ser 55 of any one of SEQID NO: 5, 6, 7, 8, 9 or 13.

In one embodiment, the amino acid pair Asp-Ser is a substituted withSer-Asp. In another embodiment, the amino acid pair Asp-Ser issubstituted with any amino acid pair other than Asn-Ser; Asn-His; orAsn-Gly. In particular embodiments, Asp is substituted with any aminoacid selected from Ala, Val, Ser, Leu, Gly or Ser. In another particularembodiment, Asp is substituted with any amino acid other than Glu orAsn. In particular embodiments, Ser is substituted with any amino acidselected from Ala, Val, Leu, Ile, Gly or Asn. In another particularembodiment, Ser is substituted with any amino acid other than Gly. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the modification is a substitution of at leastone residue selected from the residue pairs: Asn 203 and His 204; Asn315 and Gly 316; Asn 384 and Gly 385; Asn 434 and His 435 of SEQ ID NO:11. In particular embodiments, the amino acid pair is interchanged(e.g., Asn-His substituted with His-Asn; Asn-Gly substituted withGly-Asn). In another embodiment, the amino acid pair Asn-his issubstituted with any amino acid pair other than Asn-Ser; Asp-Ser; orAsn-Gly. In another embodiment, the amino acid pair Asn-Gly is asubstituted with any amino acid pair other than Asn-Ser; Asp-Ser; orAsn-His. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the amino acid modification is at a positionselected from Thr 5, Thr 20 of the light chain variable region. Inanother embodiment, the amino acid modification is at a positionselected from the group consisting of: Thr 3, Ser 7 and Thr 108 of theheavy chain constant region as set forth as SEQ ID NO: 12. In yetanother embodiment, the amino acid modification is at a positionselected from the group consisting of: Thr 120, Ser 124, Thr 225 of theheavy chain as set forth as SEQ ID NO: 13. In another embodiment, thethreonine residue is substituted with any amino acid other than serine.In another embodiment, the serine residue is substituted with any aminoacid other than threonine. In a specific embodiment, the threonineand/or serine reside is substituted with an alanine reside. Withoutwishing to be bound by any theory or mechanism of action, thesubstitution of said threonine or serine residue reduces the risk ofundesirable O-linked glycosylation.

Without wishing to be bound by any theory or mechanism of action,modifying one or both amino acid of the following amino acid pairs:Asp-Ser; Asp-Lys; Asp-Gly; reduces the risk of aspartic acidisomerization thereby preventing or decreasing the risk of degradation.In a specific embodiment, the amino acid modification is at a positionselected from the group consisting of: Asp 63-Ser 64 of SEQ ID NO: 10;Asp 169-Ser 170 of SEQ ID NO: 11; Asp 104-Lys 105, Asp 163-Gly 164, Asp282-Ser 283, Asp 285, Ser 286 of SEQ ID NO: 12; and Asp 221-Lys 222, Asp280-Gly 281, Asp 339-Ser 400, Asp 401, Ser 402 of SEQ ID NO: 13. Inparticular embodiments, the amino acid pair is interchanged (e.g.,Asp-Ser substituted with Ser-Asp). In another embodiment, the amino acidpair is substituted with any amino acid pair other than Asp-Ser, Asp-Lysor Asp-Gly. In one embodiment, Asp is substituted with Glu. Eachpossibility represents a separate embodiment of the present invention.

According to various embodiments, the antibody or antigen bindingfragment thereof of the invention has an antitumor activity of similar,or greater than, mBAT-1. According to another embodiment, said antibodyor antigen binding fragment thereof has an antitumor activity ofsimilar, or greater than, hBAT-1. According to another embodiment, saidantibody or antigen binding fragment thereof has enhanced stability ascompared to hBAT-1.

According to various embodiments, the fragment of the humanized antibodyis selected from the group consisting of: Fv, F(ab'), F(ab')2, and asingle chain antibody.

According to another embodiment, the present invention providespolynucleotide sequences encoding the antibody of the invention orfragments thereof.

According to yet another embodiment there is provided a vectorcomprising the polynucleotide sequence encoding the antibody of theinvention or fragments thereof. According to a specific embodiment thereis provided a vector comprising the polynucleotide sequence encoding theantibody of the invention or fragments thereof selected from the groupconsisting of: whole humanized antibody, the light chain variableregion, the heavy chain variable region, both chains of the variableregion.

According to another aspect, there is provided host cells containing avector comprising the polynucleotide sequence encoding the antibody ofthe invention or fragments thereof.

According to another aspect, there is provided a pharmaceuticalcomposition comprising as an active ingredient the antibody of theinvention or antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier, diluent or stabilizer.

According to another embodiment, the pharmaceutical compositioncomprising as an active ingredient the antibody of the invention is usedfor the treatment of cancer. According to another embodiment, thepharmaceutical composition may be administered either followingdetection of primary or secondary tumors in a subject, or as preventivetherapy of a subject having a high risk of developing cancers. Accordingto some embodiments, the antibody of the invention elicits anti-tumoreffects in a variety of tumors.

According to another aspect, the present invention provides a method fortreating a disease or a disorder in a subject in need thereof,comprising administering to the subject an effective amount of apharmaceutical composition comprising the antibody of the invention asan active ingredient or an antigen binding fragment thereof. In someembodiments, the disease or a disorder is cancer. In some embodiments,the disease or a disorder is an immuno-deficiency related disease ordisorder.

In one embodiment, the subject has a tumor selected from a solid tumoror a non-solid tumor. In some embodiments, the tumor is a solid tumor.In some embodiments, the tumor is a non-solid tumor. In someembodiments, the non-solid tumor is a hematologic malignancy.

In particular embodiments, the cancer is selected from the groupconsisting of a colorectal carcinoma, a non-small lung cancer (NSCLC), asmall cell lung cancer (SCLC), a breast carcinoma; a melanoma; anovarian carcinoma, a cervical carcinoma, a pancreatic cancer, a head andneck carcinoma, a gastrointestinal carcinoma, an esophageal tumor, ahepatocellular carcinoma, multiple myeloma, a renal cell carcinoma, aprostate tumor, non-Hodgkin's lymphoma, Hodgkin's disease, mantle celllymphoma, Kaposi's sarcoma, a squamous cell carcinoma, a basal cellcarcinoma, acute myeloid leukemia (AML), chronic myelocytic leukemia(CML), acute lymphocytic leukemia (ALL), and chronic lymphocyticleukemia (CLL). Each possibility represents a separate embodiment of thepresent invention.

According to specific embodiments, the cancer is selected from the groupconsisting of: colorectal carcinoma, melanoma, pancreatic cancer, headand neck carcinoma, esophageal tumor, multiple myeloma, renal cellcarcinoma, non-Hodgkin's lymphoma and Hodgkin's disease.

According to various embodiments, the subject is a non-human mammal.According to various preferred embodiments, the subject is a human.

In an additional aspect, the invention provides an antibody or antigenbinding fragment thereof of the present invention, for use in treating atumor. In another embodiment, the invention provides an antibody orantigen binding fragment thereof comprising a light chain variableregion selected from the group consisting of SEQ ID NO: 1-4, and a heavychain variable region selected from the group consisting of SEQ ID NO:5-9, wherein the antibody or antigen binding fragment thereof comprisesat least one amino acid modification at a position selected from thegroup consisting of: Thr 5, Thr 20, Cys 71, Asn 75, Ser 76, Phe 93 andPhe 97 of the light chain variable region and Asp 54, Ser 55 and Tip 107of the heavy chain variable region, for use in treating a tumor.

In an additional aspect, the invention provides an antibody or antigenbinding fragment thereof of the present invention, for the preparationof a medicament for treating a tumor. In another embodiment, theinvention provides an antibody or antigen binding fragment thereofcomprising a light chain variable region selected from the groupconsisting of SEQ ID NO: 1-4, and a heavy chain variable region selectedfrom the group consisting of SEQ ID NO: 5-9, wherein the antibody orantigen binding fragment thereof comprises at least one amino acidmodification at a position selected from the group consisting of: Thr 5,Thr 20, Cys 71, Asn 75, Ser 76, Phe 93 and Phe 97 of the light chainvariable region and Asp 54, Ser 55 and Trp 107 of the heavy chainvariable region, for the preparation of a medicament for treating atumor.

Other features and advantages of the present invention will becomeapparent from the following detailed description and appended drawingfigure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph demonstrating CD4⁺ T lymphocytes viability followingincubation with humanized monoclonal antibody BAT-1 (denoted CT-011) anda CT-011 double mutant comprising alanine substitutions at position Phe97 of the light chain variable region and Trp 107 of the heavy chainvariable region.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides humanized monoclonal antibodies,pharmaceutical compositions comprising said antibodies, and use thereoffor the treatment of a variety of indications, including but not limitedto cancer and immunodeficiency disorders. In particular, the presentinvention provides modified antibodies or fragments thereof havingspecific amino acid modifications such as, amino acid substitutions,compared to the hBAT-1 humanized monoclonal immunomodulatory antibodyvariants.

According to another embodiment, there is provided an antibody orantigen binding fragment thereof comprising a light chain variableregion at least 85% identical to any one of SEQ ID NO: 1-4, and a heavychain variable region at least 85% identical to any one of SEQ ID NO:5-9, the antibody or antigen binding fragment thereof comprises at leastone modified amino acid selected from the group consisting of: Thr 5,Thr 20, Cys 71, Asn 75, Ser 76, Phe 93 and Phe 97 in comparison with SEQID NO: 1, or at least one modified amino acid selected from the groupconsisting of: Asp 54, Ser 55 and Tip 107 in comparison with SEQ ID NO:5. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment of the invention, the antibody comprises a lightchain variable region at least 85%, at least 90%, at least 92%, at least95% or at least 98% identical to any one of SEQ ID NO: 1-4, and a heavychain variable region as set forth in any one of SEQ ID NO: 5-9. Inanother embodiment, the antibody comprises a heavy chain variable regionat least 85%, at least 90%, at least 92%, at least 95% or at least 98%identical to any one of SEQ ID NO: 5-9, and a light chain variableregion as set forth in any one of SEQ ID NO: 1-4. In another embodiment,the antibody comprises a light chain variable region at least 85%, atleast 90%, at least 92%, at least 95% or at least 98% identical to anyone of SEQ ID NO: 1-4, and a heavy chain variable region at least 85%,at least 90%, at least 92%, at least 95% or at least 98% identical toany one of SEQ ID NO: 5-9. Each possibility represents a separateembodiment of the present invention.

The term “at least X percent identical” or “having at least X percentidentity” refers to the percent of amino acid residues that areidentical in the two compared sequences when the sequences are optimallyaligned. Thus, for instance, 85% amino acid sequence identity means that85% of the amino acids in two or more optimally aligned polypeptidesequences are identical.

According to another embodiment, there is provided an antibody orantigen binding fragment thereof comprising a light chain variableregion selected from SEQ ID NO: 1-4, and a heavy chain variable regionselected from SEQ ID NO: 5-9, wherein the light chain variable regioncomprises at least one amino acid modification at a position selectedfrom the group consisting of: Thr 5, Thr 20, Cys 71, Asn 75, Ser 76, Phe93 and Phe 97; or wherein the heavy chain variable region comprises atleast one amino acid modification at a position selected from the groupconsisting of: Asp 54, Ser 55 and Trp 107. Each possibility represents aseparate embodiment of the present invention.

According to one embodiment, the light chain variable region has theamino acid sequence as set forth in SEQ ID NO: 1 (BATRK_(D)). Accordingto another embodiment, the light chain variable region has the aminoacid sequence as set forth in SEQ ID NO: 2 (BATRK_(A)). According toanother embodiment, the light chain variable region has the amino acidsequence as set forth in SEQ ID NO: 3 (BATRK_(B)). According to anotherembodiment, the light chain variable region has the amino acid sequenceas set forth in SEQ ID NO: 4 (BATRK_(C)).

According to another embodiment, the antibody or a fragment thereofcomprises at least one amino acid modification at a position selectedfrom the group consisting of: Thr 5, Thr 20, Cys 71, Asn 75, Ser 76, Phe93 and Phe 97 of any one of SEQ ID NO: 1 or SEQ ID NO: 4. Eachpossibility represents a separate embodiment of the present invention.

According to another embodiment, the antibody or a fragment thereofcomprises at least one amino acid modification at a position selectedfrom the group consisting of: Thr 5, Thr 20, Asn 75, Ser 76, Phe 93 andPhe 97 of any one of SEQ ID NO: 2 or SEQ ID NO: 3. Each possibilityrepresents a separate embodiment of the present invention.

According to another embodiment, the heavy chain variable region has theamino acid sequence as set forth in SEQ ID NO: 5 (BATRH_(C)). Accordingto another embodiment, the light chain variable region has the aminoacid sequence as set forth in SEQ ID NO: 6 (BATRH_(A)). According toanother embodiment, the light chain variable region has the amino acidsequence as set forth in SEQ ID NO: 7 (BATRH_(B)). According to anotherembodiment, the light chain variable region has the amino acid sequenceas set forth in SEQ ID NO: 8 (BATRH_(D)). According to anotherembodiment, the light chain variable region has the amino acid sequenceas set forth in SEQ ID NO: 9 (BATRH_(E)).

According to another embodiment, the antibody or antigen bindingfragment thereof of the invention comprises an amino acid modificationat a position selected from Phe 93 or Phe 97 of the light chain variableregion. In another embodiment, the light chain variable region (any oneof SEQ ID NO: 1-4) is modified by a substitution of Phe 93 or Phe 97 toany amino acid other than Trp and Tyr. Non limiting examples of aminoacid substitutions of Phe and/or Trp include Ala, Leu and Val. Inparticular embodiments, the light chain variable region comprises theamino acid sequence selected from the group consisting of: SEQ ID NO:14, 15, 16, 17, 18, 19, 20 and 21. In another particular embodiment, thelight chain variable region comprises the amino acid sequence selectedfrom the group consisting of: SEQ ID NO: 14, 16, 18, 20, and 22. In yetanother particular embodiment, the light chain variable region comprisesthe amino acid sequence selected from the group consisting of: SEQ IDNO: 15, 17, 19, 21 and 23. In an additional embodiment, the light chainvariable region comprises the amino acid sequence as set forth as SEQ IDNO: 14. In another embodiment, the light chain variable region comprisesthe amino acid sequence as set forth as SEQ ID NO: 15. In yet anotherembodiment, the Phe residue is substituted with an Ala residue.

In another embodiment, the light chain variable region comprises anamino acid substitution of Phe 93 and Phe 97 to Ala, Leu or Val. In aparticular embodiment the light chain variable region comprises has theamino acid sequence of SEQ ID NO: 22. In yet another embodiment, thelight chain variable region comprises an amino acid substitution of Phe93 and Phe 97 to Ala. In a particular embodiment the light chainvariable region comprises has the amino acid sequence of SEQ ID NO: 23.

In another embodiment of the invention, the heavy chain variable region(any one of SEQ ID NO: 5-9) is modified by a substitution of Trp 107 toany amino acid other than Phe. Non limiting examples of amino acidsubstitutions of Trp include Ala, Leu Tyr and Val. In particularembodiments, the heavy chain variable region comprises the amino acidsequence selected from the group consisting of: SEQ ID NO: 24, 25, 26,27 and 28. In another embodiment, the heavy chain variable regioncomprises the amino acid sequence as set forth as SEQ ID NO: 24. In yetanother embodiment, the Trp residue is substituted with an Ala residue.

According to another embodiment, the antibody or antigen bindingfragment thereof comprises an amino acid modification at a positionselected from Trp 107 of the heavy chain variable region and Phe 97 ofthe light chain variable region. According to yet another embodiment,the antibody or antigen binding fragment thereof comprises an amino acidmodification of Trp 107 of the heavy chain variable region and Phe 97 ofthe light chain variable region. According to another embodiment, theantibody or antigen binding fragment thereof comprises an amino acidmodification at a position selected from Trp 107 of the heavy chainvariable region and Phe 93 of the light chain variable region. Accordingto yet another embodiment, the antibody or antigen binding fragmentthereof comprises an amino acid modification of Trp 107 of the heavychain variable region and Phe 93 of the light chain variable region. Ina specific embodiment, said modification is a substitution to an alanineresidue.

TABLE 2 Exemplary variable region of hBAT-1 mutated light and heavychain comprising F93, F97 or W107 amino acid substitutions. SEQ Ig BasedID Chain on Amino acid sequence Substitution NO: Light BATRK_(D)EIVLTQSPSSLSASVGDRVTITCSARSSVS F93A, F93L 14 chainYMHWFQQKPGKAPKLWIYRTSNLASGVPSR or F93V variableFSGSGSGTSYCLTINSLQPEDFATYYCQQR region SSXPLTFGGGTKLEIK BATRK_(D)EIVLTQSPSSLSASVGDRVTITCSARSSVS F97A, F97L 15YMHWFQQKPGKAPKLWIYRTSNLASGVPSR or F97V FSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTXGGGTKLEIK BATRK_(A) EIVLTQSPSSLSASVGDRVTITCSARSSVS F93A, F93L 16YMHWYQQKPGKAPKLWIYRTSNLASGVPSR or F93V FSGSGSGTDFTLTINSLQPEDFATYYCQQRSSXPLTFGGGTKLEIK BATRK_(A) EIVLTQSPSSLSASVGDRVTITCSARSSVS F97A, F97L 17YMHWYQQKPGKAPKLWIYRTSNLASGVPSR or F97V FSGSGSGTDFTLTINSLQPEDFATYYCQQRSSFPLTXGGGTKLEIK BATRK_(B) EIVLTQSPSSLSASVGDRVTITCSARSSVS F93A, F93L 18YMHWFQQKPGKAPKLWIYRTSNLASGVPSR or F93V FSGSGSGTDYTLTINSLQPEDFATYYCQQRSSXPLTFGGGTKLEIK BATRK_(B) EIVLTQSPSSLSASVGDRVTITCSARSSVS F97A, F97L 19YMHWFQQKPGKAPKLWIYRTSNLASGVPSR or F97V FSGSGSGTDYTLTINSLQPEDFATYYCQQRSSFPLTXGGGTKLEIK BATRK_(C) EIVLTQSPSSLSASVGDRVTITCSARSSVS F93A, F93L 20YMHWFQQKPGKAPKLWIYRTSNLASGVPSR or F93V FSGSGSGTDYCLTINSLQPEDFATYYCQQRSSXPLTFGGGTKLEIK BATRK_(C) EIVLTQSPSSLSASVGDRVTITCSARSSVS F97A, F97L 21YMHWFQQKPGKAPKLWIYRTSNLASGVPSR or F97V FSGSGSGTDYCLTINSLQPEDFATYYCQQRSSFPLTXGGGTKLEIK BATRK_(D) EIVLTQSPSSLSASVGDRVTITCSARSSVS F93 & F97 22YMHWYQQKPGKAPKLWIYRTSNLASGVPSR to A, L or VFSGSGSGTDFTLTINSLQPEDFATYYCQQR SSXPLTXGGGTKLEIK BATRK_(D)EIVLTQSPSSLSASVGDRVTITCSARSSVS F93A & 23 YMHWYQQKPGKAPKLWIYRTSNLASGVPSRF97A FSGSGSGTDFTLTINSLQPEDFATYYCQQR SSAPLTAGGGTKLEIK BATRK_(D)EIVLTQSPSSLSASVGDRVTITCSARSSVS F93A 29 YMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQQR SSAPLTFGGGTKLEIK BATRK_(D)EIVLTQSPSSLSASVGDRVTITCSARSSVS F97A 30 YMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQQR SSFPLTAGGGTKLEIK Heavy BATRH_(C)QVQLVQSGSELKKPGASVKISCKASGYTFT W107A, 24 chainNYGMNWVRQAPGQGLQWMGWINTDSGESTY W107L, variableAEEFKGRFVFSLDTSVNTAYLQITSLTAED W107Y or regionTGMYFCVRVGYDALDYXGQGTLVTVSS W107V BATRH_(A)QVQLVQSGSELKKPGASVKISCKASGYTFS W107A, 25 NYGMNWVRQAPGQGLQWMGWINTDSGESTYW107L, AEEFKGRFVFSLDTSVSTAYLQITSLTAED W107Y orTGMYFCAKVGYDALDYXGQGTLVTVSS W107V BATRH_(B)QVQLVQSGSELKKPGASVKISCKASGYTFT W107A, 26 NYGMNWVRQAPGQGLQWMGWINTDSGESTYW107L, AEEFKGRFVFSLDTSVSTAYLQITSLTAED W107Y orTGMYFCAKVGYDALDYXGQGTLVTVSS W107V BATRH_(D)QIQLVQSGSELKKPGASVKISCKASGYTFT W107A, 27 NYGMNWVRQAPGQGLQWMGWINTDSGESTYW107L, AEEFKGRFVFSLDTSVNTAYLQITSLTAED W107Y orTGMYFCVRVGYDALDYXGQGTLVTVSS W107V BATRH_(E)QIQLVQSGSELKKPGASVKISCKASGYTFT W107A, 28 NYGMNWVRQAPGQGLQWMGWINTDSGESTYW107L, AEEFKGRFVFSLDTSVNTAYLQITSLTAED W107Y orTGMYFCVRVGYDALDYXGQGTLVTVSS W107V BATRH_(C)QVQLVQSGSELKKPGASVKISCKASGYTFT W107A 31 NYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAED TGMYFCVRVGYDALDYAGQGTLVTVSS

According to another embodiment, the present invention providespolynucleotide sequences encoding the antibody of the invention orfragments thereof. Accordingly, the modified antibody is produced byexpression of polynucleotides, wherein the polynucleotides may encodethe whole humanized antibody or the light chain variable region or theheavy chain variable region or the variable region of both chains of thehumanized antibody. Further, the humanized antibody may be expressed ina host cell following co-transfection of distinct vectors eachcomprising polynucleotides encoding the heavy or the light chain, or bytransfection of a single vector comprising both light and heavy chainpolynucleotide sequences.

According to yet another embodiment there is provided a vectorcomprising the polynucleotide sequence encoding the modified antibody ofthe invention or fragments thereof.

According to yet another embodiment there is provided a vectorcomprising the polynucleotide sequence encoding the modified antibody ofthe invention or fragments thereof selected from the group consistingof: whole humanized antibody, the light chain variable region, the heavychain variable region, both chains of the variable region.

According to yet another embodiment, the vector further comprises atleast one sequence encoding a component selected from the groupconsisting of: resistance genes, promoter, signal peptide, polyAtranscription terminator, selection markers, genomic human kappaconstant region.

According to yet another preferred embodiment, the components of thevector are selected from the group consisting of: Ampicillin resistancegene, Neomycin resistance gene, HCMV Immediate Early Promoter, thegenomic human kappa constant region, a mouse immunoglobulin signalpeptide sequence, Kozak sequence, a signal sequence intron, BGH polyAtranscription terminator, a Neo/G418 selection marker, a hamster dhfrselection marker.

According to yet another embodiment, the vector further comprises atleast one sequence encoding a component selected from the groupconsisting of: resistance genes, promoter, signal peptide, polyAtranscription terminator, selection markers, the genomic human Igconstant region.

According to yet another preferred embodiment, the components of thevector are selected from the group consisting of: Ampicillin resistancegene, Neomycin resistance gene, HCMV Immediate Early Promoter, thegenomic human IgG1 constant region, a mouse immunoglobulin signalpeptide sequence, Kozak sequence, a signal sequence intron, BGH polyAtranscription terminator, a Neo/G418 selection marker, a hamster dhfrselection marker.

According to another aspect, there is provided host cells containing avector comprising the polynucleotide sequence encoding the antibody ofthe invention or fragments thereof for the purposes of storage,propagation, antibody production and therapeutic applications.

According to another embodiment, the host cell may be selected from thegroup consisting of: CHO, CHO dhfr , NSO, NSO/GS, COS, COST.

Definitions

The term “modified antibodies”, “mutated antibodies” and similargrammatical expressions refer to an alteration in an amino acidsequence, for example by substitution or deletion or chemicalmodification of one or more amino acid residues, as compared to thesequence of the original, known humanized monoclonal antibodies BAT-1immunoglobulin.

The “humanized monoclonal antibodies hBAT-1” relates to a number ofhumanized monoclonal antibodies, based on murine BAT (mBAT-1), disclosedin U.S. Pat. No. 7,332,582 (the contents of which are incorporatedherein by reference). The hBAT-1 mAbs comprise a light chain variableregion selected from the group consisting of: SEQ ID NO: 1, 2, 3 and 4(denoted BATRK_(D), BATRK_(A), BATRK_(B) and BATRK_(C), respectively)and a heavy chain variable region selected from the group consisting of:SEQ ID NO: 5, 6, 7, 8 and 9 (denoted BATRH_(C), BATRH_(A), BATRH_(B),BATRH_(D) and BATRH_(E), respectively). The amino acid sequence of thelight and heavy chain antibody variants (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,8 and 9) are depicted in Table 1. The residues which differ in saidvariable regions are depicted with a gray background (positions 2, 30,69, 77, 97 and 98 of the light region and positions 35, 69, 70 and 71 ofthe heavy chain). The polynucleotide and amino-acid sequences of murineBAT (mBAT-1) are disclosed in U.S. Pat. No. 7,695,715, the contents ofwhich are incorporated herein by reference. Particular combinations of aheavy chain variable region and a light chain variable region of thehBAT-1 antibody are selected from the group consisting of: SEQ ID NO:5/SEQ ID NO: 1; SEQ ID NO: 6/SEQ ID NO: 2; SEQ ID NO: 7/SEQ ID NO: 2;SEQ ID NO: 7/SEQ ID NO: 3; SEQ ID NO: 8/SEQ ID NO: 3; and SEQ ID NO:7/SEQ ID NO: 1. In specific embodiment of the present invention, themutated antibodies are based on the above particular combinations ofheavy and light chain variable regions and comprise at least one sitespecific modification such as substitution selected from the disclosedamino acids.

An “amino acid modification” as used herein refers to an alteration inthe amino acid, for example by substitution or deletion or chemicalmodification of said amino acid. Further, the antibodies of theinvention may be chemically modified at one or more amino acid residues,either by natural processes, such as processing or otherpost-translational modifications, or by chemical modificationtechniques. Chemical modifications include, without limitation,acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPIanchor formation, covalent attachment of a liquid or lipid derivative,methylation, myristylation, pegylation, prenylation, phosphorylation,ubiqutination, or any similar process.

The term “amino acid” is used in its broadest sense to include naturallyoccurring amino acids as well as non-naturally occurring amino acidsincluding amino acid analogs. In view of this broad definition, oneskilled in the art would know that reference herein to an amino acidincludes, for example, naturally occurring proteogenic (L)-amino acids,(D)-amino acids, chemically modified amino acids such as amino acidanalogs, naturally occurring non-proteogenic amino acids such asnorleucine, and chemically synthesized compounds having properties knownin the art to be characteristic of an amino acid. As used herein, theterm “proteogenic” indicates that the amino acid can be incorporatedinto a protein in a cell through a metabolic pathway. The amino acidsused in this invention are those which are available commercially or areavailable by routine synthetic methods. Certain residues may requirespecial methods for incorporation into the peptide, and eithersequential, divergent and convergent synthetic approaches to the peptidesequence are useful in this invention. When there is no indication,either the L or D isomers may be used.

Conservative substitution of amino acids as known to those skilled inthe art are within the scope of the present invention. Conservativeamino acid substitutions includes replacement of one amino acid withanother having the same type of functional group or side chain e.g.aliphatic, aromatic, positively charged, negatively charged. One ofskill will recognize that individual substitutions, deletions oradditions to peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (Ala; A), Serine (Ser; S), Threonine (Thr; T);

2) Aspartic acid (Asp; D), Glutamic acid (Glu; E);

3) Asparagine (Asn; N), Glutamine (Gln; Q);

4) Arginine (Arg; R), Histidine (His, H), Lysine (Lys; K);

5) Isoleucine (Ile; I), Leucine (Leu; L), methionine (Met; M), Valine(Val; V); and

6) Phenylalanine (Phe; F), Tyrosine (Tyr; Y), Tryptophan (Trp; W).

According to one embodiment, the amino acid substitution is aconservative substitution.

According to another embodiment, the amino acid substitution is anon-conservative substitution. As used herein, a “non-conservativesubstitution” is any amino acid substitution other than a conservativesubstitution described above. Non limiting examples for non-conservativesubstitutions include substitution of phenylalanine or tryptophan toalanine, leucine or valine.

In a specific embodiment the amino acid modification substitutions ofthe invention (i.e., to produce the modified antibody of the invention)is other that the substitutions described in U.S. Pat. No. 7,332,582.

The term “antibody” (also referred to as an “immunoglobulin”) is used inthe broadest sense and specifically encompasses monoclonal antibodies(including full length monoclonal antibodies) and antibody fragments solong as they exhibit the desired biological activity. “Antibodyfragments” comprise a portion of a full length antibody, generally theantigen binding or variable region thereof. Examples of antibodyfragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The basic unit of the naturally occurring antibody structure is aheterotetrameric glycoprotein complex of about 150,000 daltons, composedof two identical light (L) chains and two identical heavy (H) chains,linked together by both noncovalent associations and by disulfide bonds.Each heavy and light chain also has regularly spaced intrachaindisulfide bridges. Five human antibody classes (IgG, IgA, IgM, IgD andIgE) exist, and within these classes, various subclasses, are recognizedon the basis of structural differences, such as the number ofimmunoglobulin units in a single antibody molecule, the disulfide bridgestructure of the individual units, and differences in chain length andsequence. The class and subclass of an antibody is its isotype.

The amino terminal regions of the heavy and light chains are morediverse in sequence than the carboxy terminal regions, and hence aretermed the variable domains. This part of the antibody structure confersthe antigen-binding specificity of the antibody. A heavy variable (VH)domain and a light variable (VL) domain together form a singleantigen-binding site, thus, the basic immunoglobulin unit has twoantigen-binding sites. Particular amino acid residues are believed toform an interface between the light and heavy chain variable domains(Chothia et al., J. Mol. Biol. 186, 651-63 (1985); Novotny and Haber,(1985) Proc. Natl. Acad. Sci. USA 82 4592-4596).

The carboxy terminal portion of the heavy and light chains form theconstant domains i.e. CH1, CH2, CH3, CL. While there is much lessdiversity in these domains, there are differences from one animalspecies to another, and further, within the same individual there areseveral different isotypes of antibody, each having a differentfunction.

The term “framework region” or “FR” refers to the amino acid residues inthe variable domain of an antibody which are other than thehypervariable region amino acid residues as herein defined. The term“hypervariable region” as used herein refers to the amino acid residuesin the variable domain of an antibody which are responsible for antigenbinding. The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR”. The CDRs are primarilyresponsible for binding to an epitope of an antigen. The extent of FRsand CDRs has been precisely defined (see, Kabat et al., ibid).

The term “acceptor human immunoglobulin” refers to the humanimmunoglobulin providing the framework for a humanized antibody.

As used herein, the term “humanized antibody” refers to an antibodycomprising a framework region from a human antibody and one or more CDRsfrom a non-human (usually a mouse or rat) immunoglobulin. Parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences. In some cases however, specific amino acid residues, forexample in the framework regions, may be modified, so as to optimizeperformance of the humanized antibody. Importantly, the humanizedantibody is expected to bind to the same antigen as the donor antibodythat provides the CDRs. For further details, see e.g. U.S. Pat. No.5,225,539 assigned to Medical Research Council, UK.

The terms “a framework region from an acceptor human immunoglobulin” and“a framework region derived from an acceptor human immunoblobulin”, andsimilar grammatical expressions are used interchangeably herein to referto a framework region or portion thereof that has the same amino acidsequence of the acceptor human immunoblobulin.

The term “human antibody” refers to an antibody encoded by a geneactually occurring in a human, or an allele, variant or mutant thereof.

The modified humanized monoclonal antibody of the invention ispreferably generated by recombinant DNA technology, utilizing CDRgrafting.

The term “mammal” means any mammal, including pet animals, such as dogsand cats; farm animals, such as pigs, cattle, sheep, and goats;laboratory animals, such as mice and rats; primates, such as monkeys,apes, and chimpanzees; and preferably, humans.

Methods of the Invention

Cancer immunotherapeutics are aimed by and large at modulating theresponse of the immune system to induce or enhance killing of tumorcells and control tumor growth. This approach utilizes using variousimmunomodulators including monoclonal antibodies that selectively bindto specific determinants on T cells thereby either initiating anactivation pathway or inducing an inhibitory effect.

According to another aspect, the present invention provides a method fordiagnosis or treatment of a disease or a disorder, particularly cancer,comprising administering to a subject in need thereof, an effectiveamount of a pharmaceutical composition comprising the antibody of theinvention or a fragment thereof as an active ingredient.

All types of tumors may be treated by the methods of the presentinvention. The tumors may be solid or non-solid.

Some examples of solid tumors that can be treated with the combinationof the present invention include carcinomas, sarcomas, blastomas orgliomas. Some examples of such tumors include epidermoid tumors,squamous tumors, such as head and neck tumors, colorectal tumors,prostate tumors, breast tumors, lung tumors, including small cell andnon-small cell lung tumors, pancreatic tumors, thyroid tumors, ovariantumors, liver tumors, esophageal tumors and gastric tumors. Otherexamples include Kaposi's sarcoma, CNS neoplasms, neuroblastomas,capillary hemangioblastomas, meningiomas and cerebral metastases,melanoma, gastrointestinal and renal carcinomas and sarcomas,rhabdomyosarcoma, glioblastoma, preferably glioblastoma multiforme, andleiomyosarcoma. Examples of vascularized skin cancers include squamouscell carcinoma, basal cell carcinoma and skin cancers that can betreated by suppressing the growth of malignant keratinocytes, such ashuman malignant keratinocytes.

Some examples of non-solid tumors include leukemias, multiple myelomasand lymphomas. Some examples of leukemias include acute myelocyticleukemia (AML), chronic myelocytic leukemia (CML), acute lymphocyticleukemia (ALL), chronic lymphocytic leukemia (CLL), erythrocyticleukemia or monocytic leukemia. Some examples of lymphomas includelymphomas associated with Hodgkin's disease, Non-Hodgkin's disease ormantle cell lymphoma.

Currently preferred types of tumors are selected from the followinggroup: colorectal carcinoma; lung carcinoma including non-small lungcancer (NSCLC) and small cell lung cancer (SCLC); breast carcinoma;melanoma; ovarian carcinoma; cervical carcinoma, pancreatic cancer; headand neck carcinoma; gastrointestinal carcinoma; esophageal tumors;hepatocellular carcinoma; multiple myeloma; renal cell carcinoma;prostate tumors; non-Hodgkin's lymphoma; Hodgkin's disease; mantle celllymphoma; Kaposi's sarcoma; squamous cell carcinoma; basal cellcarcinoma; acute myeloid leukemia (AML); chronic myelocytic leukemia(CML); acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia(CLL).

The term “antitumor effect” as used herein, refers to a beneficialbiological effect, which can be manifested by any one or more of: adecrease or stabilization of tumor volume, a decrease or stabilizationof the number of tumor cells, a decrease or stabilization of the rate oftumor growth, a decrease or stabilization of the number of metastases,protection from tumor recurrence, an increase in life expectancy orsurvival of the subject with the tumor, an increase in life expectancyor survival without disease progression of the subject with the tumor oramelioration of various physiological symptoms associated with thecancerous condition. An “antitumor effect” can also be manifested by theability to prevent the occurrence of tumor in the first place or therecurrence of the tumor. Given its properties, the methods of theinvention can be used in the treatment of acute cancer, of dormant,controlled or stabilized cancer, as well as in cancer prophylaxis.

The term “enhanced survival”, as used herein, refers to a prolongedlength of time during which the subject or patient is alive followingtreatment with a method of the invention. Enhanced survival denotes theincreased probability of staying free of disease progression for anindividual suffering from cancer after a particular treatment. It isalso used to describe the elevated percentage of individuals in a groupwhose disease is likely to remain stable (not showing signs ofprogression) after a specified duration of time, compared to a controlgroup. It is also used to describe the elevated percentage ofindividuals in a group whose disease is likely to be cured (not showingsigns of disease) after a specified duration of time, compared to acontrol group. This parameter may be measured by any one of thecustomary clinical endpoints denoted as “progression-free survival”,“overall survival” and “disease free survival” used as an indication ofthe efficacy of a particular treatment.

The term “tumor recurrence” refers to the re-emergence, reappearance,re-growth or proliferation of a tumor of the same type in either thesame location or a different location, following a period during whichthe growth of the original tumor has been reversed, arrested orinhibited.

The term “enhances or increases lymphocyte survival” as used hereinrefers to the ability of the antibodies of the invention to prolong theviability of lymphocytes in vitro or in vivo, as compared to theviability of an identical cell population not contacted with theantibody of the invention. With regard to the combination therapy of theinvention, he term refers to the ability of a particular combination oftreatments to prolong the viability of lymphocytes in vitro or in vivo,as compared to the viability of an identical cell population with onlyone of the treatments.

An antitumor activity of similar to mBAT-1 or hBAT-1 refers to anantitumor effect of about 5%, or not more than 10%, similar to theantitumor effect of mBAT-1 or hBAT-1. An antitumor activity of greaterthan mBAT-1 or hBAT-1 refers to an antitumor activity of more than 10%,more than 30% or more than 30%, as compared to the antitumor activity ofgreater than mBAT-1 or hBAT-1.

According to another embodiment, the antibody of the invention inadministered together with, prior to, or following, the administrationof other agents, which can act in an additive or synergistic manner withit.

According to yet another embodiment, the antibody of the invention inadministered together with, prior to, or following, the administrationof agents selected from the group consisting of: cytokines, IL-1(Interleuken-1), IL-2, IL-6, IFN-α (Interferon-α), cell vaccines,antibodies, T-cell stimulatory antibodies, anti-tumor therapeuticantibodies. Each possibility is a separate embodiment of the presentinvention.

In another embodiment, the disease or a disorder treated by the methodsof the invention is an immuno-deficiency related disease or disorderselected from severe combined immunodeficiency disease, acquiredimmunodeficiency syndrome, and any disorder that involves depletion,attenuation and/or malfunctioning of lymphocytes, specifically T cells,NK cells, NK-T cells, B cells, monocytes, macrophages or any combinationthereof.

In another embodiment, the disease or a disorder is an immunodeficiency,immune malfunction or immune incompetence, collectively referred tohereinafter as immunodeficiency disorders, established afterchemotherapy or irradiation. According to certain embodiments, thepharmaceutical composition comprising the antibody of the invention isused in conjunction with autologous, allogeneic or syngeneic stem celltransplantation derived from the bone marrow, umbilical cord blood orperipheral blood and donor leukocyte infusion.

According to yet another embodiment, the immunodeficiency disorder isselected from the group consisting of: severe combined immunodeficiencydisease, acquired immunodeficiency syndrome, X-linkedagammaglobulinaemia, common variable immunodeficiency, IgA deficiency,IgG subclass deficiency, Wiskott-Aldrich syndrome, DiGeorge anomaly,Ataxia Telangiectasia, adenosine deaminase deficiency andactivation-induced cytidine deaminase deficiency.

According to yet another embodiment, the immunodeficiency disorder isrelated to viral infection, fungal infection or bacterial infection.According to yet another embodiment, the immunodeficiency disorder isassociated with intoxication.

According to yet another embodiment, the method of the invention is usedfor treating anemia, particularly, aplastic anemia and myelodysplasticsyndromes (MDS), primarily for avoiding further complication of theanemia due to immunodeficiency disorders.

Combination Therapy with Chemotherapy

According to certain aspects of the present invention, administration ofthe immunostimulatory humanized antibody comprising modified amino acidsin conjunction with at least one antitumor chemotherapeutic agent actsto enhance the antitumor effect of chemotherapeutic agents, and viceversa. In preferred embodiments, the combinations of theimmunostimulatory antibody together with the at least onechemotherapeutic agent improve the clinical outcome in a significantmanner versus each of the treatments alone. In a preferred embodiment,there is synergy when tumors are treated with the humanized antibody ofthe invention in conjunction with at least one chemotherapeutic agent,and, optionally further in conjunction with radiation.

In other words, according to one aspect of the present invention theantitumor effect of the humanized antibody of the invention is augmentedmore than expected when combined with at least one chemotherapeuticagent.

Antitumor effect induced by the combinations of the invention includesthe prevention, inhibition of the progression of a tumor, reduction oftumor growth and protection against tumor recurrence, includingcancerous and noncancerous tumors. The progression of a tumor includesthe invasiveness, metastasis, recurrence and increase in size of thetumor. The reduction of tumor growth also includes the destruction orelimination of a tumor leading to complete remission.

In another embodiment, the antibody of the invention is effective forimproving tolerability to chemotherapeutic agents. As is known in theart, a major setback for patients undergoing cancer chemotherapy is theappearance of severe and detrimental adverse side effects due to thepotent toxicity of most chemotherapeutic agents.

The invention further provides a method of enhancing survival in asubject with a tumor, which comprises administration of the humanizedantibody of the invention, either on its own, or optionally, combinedwith the further administration of one or more chemotherapeutic agents.

The invention further provides a method of reducing or preventingrecurrence of a tumor, which comprises administration of the humanizedantibody of the invention, either on its own, or optionally, combinedwith the further administration of one or more chemotherapeutic agents.In some embodiments, combination treatment of experimental animals usingthe humanized antibody of the invention and chemotherapeutic agentsinduced a “memory” effect, such that tumor recurrence is inhibited uponre-challenge with the original tumor type.

According to another embodiment, the present invention provides a methodof treating a tumor, the method comprising (i) administering to asubject in need thereof an effective amount of an antibody of theinvention or a fragment thereof; and (ii) administering to the subjectan effective amount of at least one chemotherapeutic agent; therebytreating the tumor.

According to another embodiment, the invention further provides a methodof improving tolerability to at least one chemotherapeutic agent, themethod comprising administering to a subject in need thereof aneffective amount of an antibody of the invention or a fragment thereof,wherein the subject is undergoing chemotherapy with at least onechemotherapeutic agent; thereby improving tolerability to saidchemotherapeutic agent.

The term “tolerability to chemotherapeutic agents” refers to thephysiological, physicochemical and immunological capacity of a subjectto tolerate the adverse side effects associated with treatment with oneor more chemotherapeutic agents. Accordingly, the term “improvingtolerability to chemotherapeutic agents” refers to increasing thephysiological and physicochemical capacity to such adverse side effects,such that the severity of the adverse side effects is decreased and/orthe number of side effects is decreased. Accordingly, “improvingtolerability to chemotherapeutic agents” may refer to improving thequality of life of cancer patients treated with chemotherapeutic agents.

According to yet another embodiment of the invention there is provided amethod of enhancing survival or inhibiting disease progression in asubject having a tumor, wherein the subject is treated with at least onechemotherapeutic agent, the method comprising administering an effectiveamount of an antibody of the invention or a fragment thereof, therebyenhancing survival of the subject.

According to yet another embodiment, the invention provides a method ofreducing or preventing tumor recurrence, the method comprisingadministering to a subject in need thereof an effective amount of anantibody of the invention or a fragment thereof, thereby reducing orpreventing tumor recurrence. According to one embodiment, the method ofreducing or preventing tumor recurrence further comprises administeringto the subject at least one chemotherapeutic agent. According toparticular embodiments, the subject is undergoing or has completed acourse of chemotherapy with at least one chemotherapeutic agent.

According to various embodiments, the administering of the antibody ofthe invention and of the at least one chemotherapeutic agent is carriedout substantially simultaneously, concurrently, alternately,sequentially or successively. In some embodiments, the antibody and theat least one chemotherapeutic agent are administered according tooverlapping schedules.

According to particular embodiments, administering of the antibody iscarried out prior to initial administration of the at least onechemotherapeutic agent.

According to other embodiments, administering of either or both of theantibody and the at least one chemotherapeutic agent is carried out by aroute selected from the group consisting of intravenous, oral,intraperitoneal, subcutaneous, isolated limb perfusion, infusion into anorgan and combinations thereof.

In particular embodiments, the methods of the invention further compriseassessing at least one parameter selected from the group consisting of:rate of tumor growth, tumor volume, number of metastases, tumorrecurrence and combinations thereof.

It should be noted that according to the teaching of the presentinvention, the modified humanized antibody of the invention may beadministered before, during, or after commencing chemotherapy and,optionally, radiation therapy, as well as any combination thereof, i.e.before and during, before and after, during and after, or before,during, and after commencing the chemotherapy and, optionally, theradiation therapy. For example, the antibody of the invention may beadministered between 1 and 30 days prior to or after commencingchemotherapy. The antibody may further be administered between coursesof chemotherapy.

In the combination therapy methods of the invention, the antibodies maybe administered in parallel to the chemotherapy, for examplesubstantially simultaneously or concurrently. Other administrationschedules may also be used, for example, overlapping schedules or thosewhich involve alternately, sequentially or successively administeringthe two types of treatment.

According to various embodiments, the at least one chemotherapeuticagent is selected from the group consisting of: antimetabolites,platinum-based drugs, mitotic inhibitors, anthracycline antibiotics,topoisomerase inhibitors, anti-angiogenic agents and combinationsthereof.

According to another particular embodiment, the at least onechemotherapeutic agent is selected so that the modified hBAT-1 of theinvention enhances survival of lymphocytes when used in combination withthe chemotherapeutic agent. Typically, the enhanced or increasedsurvival may be conveniently assayed in vitro.

Accordingly, in various embodiments, the chemotherapeutic agent may beselected from an antimetabolite, such as the pyrimidine analog 5-fluorouracil, or cytarabin, or a platinum-based drug, such asoxaliplatin or cisplatin. Further, in various embodiments, thechemotherapeutic agent may be other than an agent selected from atopoisomerase I inhibitor (such as SN-38) and an alkylating agent (suchas cyclophosphamide).

According to some embodiments, the at least one chemotherapeutic agentis an antimetabolite, including purine antagonists, pyrimidineantagonists and folate antagonists. According to some embodiments, theantimetabolite is a pyrimidine antagonist. According to someembodiments, the antimetabolite is selected from the group consistingof: 5 -fluorouracil, uracil mustard, uracil, capecitabine,6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine,and pemetrexed.

According to some embodiments, the at least one chemotherapeutic agentis 5-fluorouracil. According to some embodiments, the at least onechemotherapeutic agent is cytarabine. According to some embodiments, theat least one chemotherapeutic agent is a platinum-based drug selectedfrom the group consisting of: cisplatin, carboplatin and oxaliplatin.According to yet other embodiments, the at least one chemotherapeuticagent is a mitotic inhibitor selected from the group consisting of:paclitaxel, docetaxel, etoposide, vinblastine, vincristine andvinorelbine. According to yet other embodiments, the at least onechemotherapeutic agent is an anthracycline antibiotic selected from thegroup consisting of: daunorubicin, respinomycin D and idarubicin.According to some embodiments, the at least one chemotherapeutic agentis an anti-angiogenic agent selected from the group consisting of:bevacizumab, dopamine, tetrathiomolybdate, and antiangiogenic variantsof VEGF. According to some embodiments, the at least onechemotherapeutic agent is other than a topoisomerase I inhibitor.According to some embodiments, the at least one chemotherapeutic agentis other than an alkylating agent.

Chemotherapy drugs are divided into several groups based on their effecton cancer cells, the cellular activities or processes the druginterferes with, or the specific phases of the cell cycle the drugaffects. Accordingly, chemotherapy drugs fall in one of the followingcategories: alkylating agents, nitrosoureas, antimetabolites,anthracyclines, topoisomerase I and II inhibitors, mitotic inhibitors,inter alia platinum based drugs, steroids and anti-angiogenic agents.

Antimetabolites, also termed “nucleoside analogs”, replace naturalsubstances as building blocks in DNA molecules, thereby altering thefunction of enzymes required for cell metabolism and protein synthesis.In the event that they mimic nutrients required for cell growth, thecells eventually undergo lysis. If a nucleoside is replaced with anon-functional nucleoside analog, the latter is incorporated into DNAand RNA, finally inducing cell cycle arrest and apoptosis by inhibitingthe cell's ability to synthesize DNA. Antimetabolites are cell-cyclespecific and are most effective during the S-phase of cell division asthey primarily act upon cells undergoing synthesis of new DNA forformation of new cells. The toxicities associated with these drugs areseen in cells that are growing and dividing quickly. Examples ofantimetabolites include purine antagonists, pyrimidine antagonists, andfolate antagonists. These agents damage cells during the S phase and arecommonly used to treat leukemias, tumors of the breast, ovary, and thegastrointestinal tract, as well as other cancers. Specific examples ofantimetabolites include 5-fluorouracil (also known as 5FU),capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine,fludarabine and pemetrexed.

Platinum-based chemotherapeutic drugs crosslink DNA in several differentways, interfering with cell division by mitosis. The damaged DNA elicitsDNA repair mechanisms, which in turn activate apoptosis when repairproves impossible. Most notable among the DNA changes are the1,2-intrastrand cross-links with purine bases. These include1,2-intrastrand d(GpG) adducts which form nearly 90% of the adducts andthe less common 1,2-intrastrand d(ApG) adducts. 1,3-intrastrand d(GpXpG)adducts occur but are readily excised by the nucleotide excision repair(NER). Other adducts include inter-strand crosslinks and nonfunctionaladducts that have been postulated to contribute to the activity ofplatinum-based drugs. Interaction with cellular proteins, particularlyHMG domain proteins, has also been advanced as a mechanism ofinterfering with mitosis, although this is probably not its primarymethod of action. Platinum-based chemotherapeutic drugs includecisplatin (also known as cisplatinum or cis-diamminedichloridoplatinumII (CDDP), carboplatin and oxaliplatin. Cisplatin is frequentlydesignated as an alkylating agent, though it has no alkyl group andcannot carry out alkylating reactions. It is correctly classified asalkylating-like. Platinum-based chemotherapeutic drugs are used to treatvarious types of cancers, including sarcomas, some carcinomas (e.g.small cell lung cancer, and ovarian cancer), lymphomas and germ celltumors.

Mitotic inhibitors interfere with cell division. The most knownchemotherapeutic agent in this category is paclitaxel (also known asTaxol®, “plant alkaloid”, “taxane” and an “antimicrotubule agent”).Together with docetaxel, it forms the drug category of the taxanes.However, other mitotic inhibitors are known, including, but not limitedto etoposide, vinblastine and vincristine. Paclitaxel acts byinterfering with normal microtubule growth during cell division byarrests their function; it hyper-stabilizes their structure. Thisdestroys the cell's ability to use its cytoskeleton in a flexiblemanner. Specifically, paclitaxel binds to the β subunit of tubulin, the“building block” of microtubules, and the binding of paclitaxel locksthese building blocks in place. The resulting microtubule/paclitaxelcomplex does not have the ability to disassemble. This adversely affectscell function because the shortening and lengthening of microtubules(termed dynamic instability) is necessary for their function as amechanism to transport other cellular components. For example, duringmitosis, microtubules position the chromosomes all through theirreplication and subsequent separation into the two daughter-cell nuclei.Furthermore, paclitaxel induces programmed cell death (apoptosis) incancer cells by binding to the apoptosis stopping protein Bc1-2 (B-cellleukemia 2) and thus arresting its function.

Another group of DNA-interacting drugs widely used in anti-cancerchemotherapy is the group of anthracycline antibiotics which includes,inter alia, daunorubicin, doxorubicin (also known as Adriamycin® anddoxorubicin hydrochloride), respinomycin D and idarubicin. These drugsinteract with DNA by intercalation and inhibition of macromolecularbiosynthesis thereby inhibiting the progression of the enzymetopoisomerase II, which unwinds DNA for transcription. They stabilizethe topoisomerase II complex after it has broken the DNA chain forreplication, preventing the DNA double helix from being resealed andthereby stopping the process of replication. It is commonly used in thetreatment of a wide range of cancers.

Alkylating antineoplastic agents directly attack DNA. They attach analkyl group to DNA, cross-linking guanine nucleobases in DNAdouble-helix strands. This makes the strands unable to uncoil andseparate. As this is necessary in DNA replication, the cells can nolonger divide. These drugs act nonspecifically. Cyclophosphamide is analkylating agent, however, it is a highly potent immunosuppressivesubstance.

Topoisomerase I and II inhibitors interfere with the enzymatic activityof topoisomerase I and 2, respectively, eventually leading to inhibitionof both DNA replication and transcription. Examples of topoisomerase Iinhibitors include topotecan and irinotecan. Irinotecan, is a prodrugconverted to a biologically active metabolite7-ethyl-10-hydroxy-camptothecin (SN-38) by a carboxylesterase-convertingenzyme. One thousand-fold more potent than its parent compoundirinotecan, SN-38 inhibits topoisomerase I activity by stabilizing thecleavable complex between topoisomerase I and DNA, resulting in DNAbreaks that inhibit DNA replication and trigger apoptotic cell death.Because ongoing DNA synthesis is necessary for irinotecan to exert itscytotoxic effects, it is also classified as an S-phase-specific agent.Examples of topoisomerase II inhibitors include etoposide andteniposide.

Anti-angiogenic agents interfere with the generation of new bloodvessels, eventually leading to the “starvation” of tumors. Non-limitingexamples of anti-angiogenic agents include the monoclonal antibodybevacizumab, dopamine and tetrathiomolybdate.

Vascular endothelial growth factor (VEGF) is a 32-42 kDa dimericglycoprotein which mediates vasodilatation, increased vascularpermeability and endothelial cell mitogenesis. Differential exonsplicing of the VEGF gene results in three main mRNA species which codefor three secreted isoforms (subscripts denote numbers of amino acids):VEGF189, VEGF165, and VEGF121. A number of minor splice variants havealso been described (VEGF206, VEGF183, VEGF145 and VEGF148). Variants ofVEGF polypeptides and their use in cancer therapy is disclosed forexample, in WO/2003/012105.

Combination Therapy with Radiation

According to various embodiments, the methods further comprise treatingthe subject with radiation. According to various embodiments, themethods comprise all of administering the antibody of the invention,administering the at least one chemotherapeutic agent and treating thesubject with radiation. According to some embodiments, the antibody, theat least one chemotherapeutic agent and radiation treatment areadministered substantially simultaneously, concurrently, alternately,successively or according to overlapping schedules.

The source of radiation that may be used in combination with themodified antibody of the invention and the chemotherapeutic agent(s) canbe either external or internal to the patient being treated. When thesource is external to the patient, the therapy is known as external beamradiation therapy (EBRT). When the source of radiation is internal tothe patient, the treatment is called brachytherapy (BT).

Radiation is administered in accordance with well-known standardtechniques using standard equipment manufactured for this purpose, suchas AECL Theratron and Varian Clinac.

The distance between the source of the external radiation and the pointof entry into the patient may be any distance that represents anacceptable balance between killing target cells and minimizing sideeffects. Typically, the source of the external radiation is between 70and 100 cm from the point of entry into the patient.

Brachytherapy is generally carried out by placing the source ofradiation in the patient. Typically, the source of radiation is placedapproximately 0-3 cm from the tissue being treated. Known techniquesinclude interstitial, intercavitary, and surface brachytherapy. Theradioactive seeds can be implanted permanently or temporarily. Sometypical radioactive atoms that have been used in permanent implantsinclude iodine-125 and radon. Some typical radioactive atoms that havebeen used in temporary implants include radium, cesium-137, andiridium-192. Some additional radioactive atoms that have been used inbrachytherapy include americium-241 and gold-198.

The dose of radiation depends on numerous factors as is well known inthe art. Such factors include the organ being treated, the healthyorgans in the path of the radiation that might inadvertently beadversely affected, the tolerance of the patient for radiation therapy,and the area of the body in need of treatment. The dose will typicallybe between 1 and 100 Gy, and more particularly between 2 and 80 Gy. Somedoses that have been reported include 35 Gy to the spinal cord, 15 Gy tothe kidneys, 20 Gy to the liver, and 65-80 Gy to the prostate. It shouldbe emphasized, however, that the invention is not limited to anyparticular dose. The dose will be determined by the treating physicianin accordance with the particular factors in a given situation,including the factors mentioned above.

The dose of radiation for brachytherapy can be the same as thatmentioned above for external beam radiation therapy. In addition to thefactors mentioned above for determining the dose of external beamradiation therapy, the nature of the radioactive atom used is also takeninto account in determining the dose of brachytherapy.

Humanized Antibody of the Invention

As used herein, the terms “BAT” and a BAT antibody” are used in a broadsense and specifically cover antibodies identical to or based on themurine monoclonal antibody known as mBAT-1, or an antigen bindingfragment thereof. The monoclonal antibody mBAT-1 is secreted by thehybridoma cell line deposited at the Collection Nationale de Cultures deMicroorganismes (CNCM), under Accession No. 1-1397, as disclosed in U.S.Pat. No. 5,897,862. Further “BAT” and a BAT antibody” may refer to anantibody, which recognizes the same antigenic epitope as mBAT-1, forexample a chimeric antibody as described in U.S. Patent ApplicationPublication No. 2003/0026800. A BAT antibody also includes a humanizedantibody, various examples of which are disclosed in WO03/099196 andU.S. Patent Application Publication No. 2008/0025980 and interchangeablydenoted “CT-011”, “hBAT” and “hBAT-1”.

In general, the light chain variable region of the humanized monoclonalantibody is characterized by the formula:

FR_(L1)—CDR_(L1)—FR_(L2)—CDR_(L2)—FR_(L3)—CDR_(L3)—FR_(L4)

wherein each FR is independently a framework region of a human antibodyand each CDR is independently a complementarity determining region ofthe monoclonal mBAT-1 antibody.

In general, the heavy chain variable region of the humanized monoclonalantibody is characterized by the formula:

FR_(H1)—CDR_(H1)—FR_(H2)—CDR_(H2)—FR_(H3)—CDR_(H3)—FR_(H4)

wherein each FR is independently a framework region of a human antibodyand each CDR is independently a complementarity determining region ofthe monoclonal mBAT-1 antibody.

In particular embodiments, the FRs are derived from the light chainvariable region of the human TEL9 antibody, or are modified therefrom incertain amino acid residues. Human TEL-9 antibody was identified indiverse libraries of immunoglobulin heavy (VH) and light (V kappa and Vlambda) chain variable (V) genes prepared from peripheral bloodlymphocytes of unimmunized donors (Marks et al. J Mol Biol. 1991,222:581-97). This antibody was shown to bind specifically to the turkeyegg-white lysozyme (TEL) antigen.

In particular embodiments, the FRs are derived from the heavy chainvariable region of the human hsighv1295 antibody, or modified therefromin certain amino acid residues. Human hsiggv1295 antibody was isolatedfrom stable hybridomas and Epstein-Barr virus-transformed B cell linesfrom the synovial fluid or peripheral blood of three patients withrheumatoid arthritis and one patient with systemic lupus erythematosus(Fang et al., J Exp Med. 1994, 179:1445-56).

Compositions, Administration and Dosages

For use in the methods of the invention, the humanized antibody may beformulated in a conventional manner using one or more pharmaceuticallyacceptable carriers, stabilizers or excipients (vehicles) to form apharmaceutical composition as is known in the art, in particular withrespect to protein active agents. Carrier(s) are “acceptable” in thesense of being compatible with the other ingredients of the compositionand not deleterious to the recipient thereof. Suitable carrierstypically include physiological saline or ethanol polyols such asglycerol or propylene glycol.

The antibody may be formulated as neutral or salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups) and which are formed with inorganicacids such as hydrochloric or phosphoric acids, or such organic acidssuch as acetic, oxalic, tartaric and maleic. Salts formed with the freecarboxyl groups may also be derived from inorganic bases such as sodium,potassium, ammonium, calcium, or ferric hydroxides, and organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine andprocaine.

The compositions may be suitably formulated for intravenousintramuscular, subcutaneous, or intraperitoneal administration andconveniently comprise sterile aqueous solutions of the antibody, whichare preferably isotonic with the blood of the recipient. Suchformulations are typically prepared by dissolving solid activeingredient in water containing physiologically compatible substancessuch as sodium chloride, glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be prepared in unit ormulti-dose containers, for example, sealed ampoules or vials.

The compositions may incorporate a stabilizer, such as for examplepolyethylene glycol, proteins, saccharides (for example trehalose),amino acids, inorganic acids and admixtures thereof. Stabilizers areused in aqueous solutions at the appropriate concentration and pH. ThepH of the aqueous solution is adjusted to be within the range of5.0-9.0, preferably within the range of 6-8. In formulating theantibody, anti-adsorption agent may be used. Other suitable excipientsmay typically include an antioxidant such as ascorbic acid.

The compositions may be formulated as controlled release preparationswhich may be achieved through the use of polymer to complex or absorbthe proteins. Appropriate polymers for controlled release formulationsinclude for example polyester, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, and methylcellulose. Another possible method forcontrolled release is to incorporate the antibody into particles of apolymeric material such as polyesters, polyamino acids, hydrogels,poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively,instead of incorporating these agents into polymeric particles, it ispossible to entrap these materials in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macro emulsions.

When oral preparations are desired, the compositions may be combinedwith carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic.

The humanized antibody of the invention is preferably administeredparenterally, generally by intravenous infusion. Administration may alsobe by intraperitoneal, oral, subcutaneous, or intramuscular routes.Antibodies are generally administered in the range of about 0.1 to about20 mg/kg of patient weight, commonly about 0.5 to about 10 mg/kg, andoften about 1 to about 5 mg/kg. In this regard, it is preferred to useantibodies having a circulating half-life of at least 12 hours,preferably at least 4 days, more preferably up to 21 days. Chimeric andhumanized antibodies are expected to have circulatory half-lives of upto four and up to 14-21 days, respectively. In some cases it may beadvantageous to administer a large loading dose followed by periodic(e.g., weekly) maintenance doses over the treatment period. Antibodiescan also be delivered by slow-release delivery systems, pumps, and otherknown delivery systems for continuous infusion. Dosing regimens may bevaried to provide the desired circulating levels of a particularantibody based on its pharmacokinetics. Thus, doses will be calculatedso that the desired circulating level of therapeutic agent ismaintained.

Typically, the effective dose will be determined by the condition of thesubject, as well as the body weight or surface area of the subject to betreated. The size of the dose and the dosing regimen also will bedetermined by the existence, nature, and extent of any adverse sideeffects that accompany the administration of modified antibodies of theinvention in a particular subject. In determining the effective amountof the therapeutic composition to be administered, the physician needsto evaluate inter alia circulating plasma levels, toxicity, andprogression of the disease.

The term “effective amount” with respect to the humanized antibody andthe chemotherapeutic agent(s) of the invention should be understood asmeaning an amount of each of these active agents required to achieve atherapeutic effect, without causing excessive or uncontrollable adverseside effects. The effective amount required to achieve the therapeuticend result may depend on a number of factors including, for example, thespecific type of the tumor and the severity of the patient's condition,and whether the combination is further co-administered with radiation.The effective amount (dose) of the active agents, in the context of thepresent invention should be sufficient to effect a beneficialtherapeutic response in the subject over time, including inhibition oftumor growth, reduction in the rate of tumor growth, prevention of tumorand metastasis growth and enhanced survival.

In various embodiments of the combination methods of the invention, theantibody and the chemotherapeutic agent may be administered according toany of a number of treatment schedules, also referred to “dosingschedules” and “administration regimens”, referring to the frequency ofadministration and order of administration of each active agent. Forexample, the antibody and the chemotherapeutic agent may be administeredsubstantially simultaneously i.e. at the same time, using for example acombined dosage form or separate dosage forms. This form ofadministration may also be referred to as “concomitant” administration.Concurrent administration refers to administration of the active agentswithin the same general time period, for example on the same day(s) butnot necessarily at the same time. For example, one active agent mayrequire administration with food, while the other requiresadministration in the semi-fasting state. Alternate administrationincludes administration of one agent during a particular time period,for example over the course of a few days or a week, followed byadministration of the other agent during a subsequent identical periodof time, and then repeating the pattern for one or more cycles.Sequential or successive administration includes administration of oneagent during a first time period, using one or more doses, followed byadministration of the other agent during a second time period using oneor more doses. An overlapping schedule may also be employed, whichincludes administration of the active agents on different days over thetreatment period, not necessarily according to a regular sequence.Variations on these general guidelines may also be employed, accordingto the agents used and the condition of the subject.

In some particular combinations, it may be advantageous to use aspecific sequence of administration e.g. one agent prior to the other.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention.

EXAMPLES Example 1 CD4^(′) T Lymphocytes Viability Following Incubationwith a CT-011 Double Mutant

A CT-011 double mutant antibody comprising amino acid substitutions ofTrp 107 to Ala and Phe 97 to Ala was formed (denoted w222+F97).Representative example of 48 hours activated human CD4⁺ T lymphocytesincubated with 1 ug/ml or 0.1 ug/ml CT-011 or CT-011 double mutantW222+F97 or isotype control antibody for additional 72 hours. Cellviability was evaluated by Trypan Blue exclusion.

As seen in FIG. 1, lymphocyte viability is higher at the culturesincluding the double mutant antibody. Y-axis of FIG. 1 indicatesconcentration of viable cells.

Example 2 In Vivo Effect of the Modified hBAT-1 in a Murine Tumor Model

To examine whether the modified antibodies of the present invention cantransmit the biological effects characteristic of mBAT-1 or hBAT-1, theefficacy of the modified antibodies is studied in vivo.

C57BL mice are inoculated with B16 melanoma cells to induce lungmetastases. Increasing amounts (1, 10 and 20 μg) of a modified mAb ofthe invention is injected on day 12 post tumor-inoculation and comparedto an optimal dose of 10 μg mBAT-1 and/or hBAT-1. Lung weight may bemeasured on Day 24 post tumor inoculation to indicate the establishmentof a tumor. The average lung weight per treatment is indicative of thevarious tested mAbs.

Example 3 Inhibition of Human Melanoma (SK-28) in SCID Mice by theModified hBAT-1

Mouse and human BAT-1 mAb have been shown to inhibit the formation ofhuman-tumor metastases in the presence of human peripheral bloodlymphocytes (hPBL). To estimate the efficacy of modified hBAT-1 ininhibition of human cancer, the modified antibody is studied in a modelcombining both tumors and lymphocytes of human origin. Severe combinedimmune-deficient mice (SCID) is engrafted with hPBL to restoreimmune-competence. Mice are challenged with human melanoma cells (SK-28)and treated with increasing concentrations of the humanized antibody,administered in a single i.v. dose on day 11 post tumor inoculation.

Example 4 Immunotherapy of Human Colorectal Cancer Hepatic Metastases bythe Modified hBAT-1 mAb in Nude Mice

LIM6 and HM7 are two sub-clones of the human CRC cell line LS174T thatwere selected for their high mucin synthesis and metastatic potential.The tumor cells are injected into the exposed spleen of anesthetizednude mice. After 1 minute, the spleens are removed and the excisionsclosed. Low doses of mBAT-1, hBAT-1 and modified hBAT-1 antibodies ofthe invention are administered 12 days later and mice are sacrificed 35days post tumor inoculation. The livers are weighed, the number ofmetastatic nodules counted, and liver tissue processed for histology andImmunohistochemistry study.

Example 5 Co-Localization of the Modified hBAT with CD4 and CD8

Mouse and human BAT-1 have been shown to bind human lymphocytes,recognizing both CD4+ and CD8+ subsets. To establish the bindingspecificity of the modified humanized mAbs of the invention, humanPeripheral Blood Lymphocytes (PBL) are isolated from the blood of normaldonors, as described hereinbelow, and analyzed for co-localization ofhBAT with known lymphocyte markers.

Peripheral blood mononuclear cells (PBMC) are isolated by ficoll andincubated in tissue culture plates to remove adherent cells. Isolatedare were gated on lymphocytes by size and granularity and on live cellsby propidium iodine (PI) exclusion. Binding is performed at 4° C. for 1hr, and determined by flow cytometry on gated lymphocytes.

Example 6 Binding of the Modified hBAT-1 to B Lymphocytes

The modified humanized mAb of the invention are raised against themembranes of Daudi cells, a human B lymphoma cell-line. PBL from normaldonors are isolated by ficoll, as known in the art, followed byadherence to tissue culture plates. Non-adherent cells are examined forthe co-localization of the antibodies with B-cell markers including CD19and CD20. Binding is performed at 4° C. for 1 hr, and determined by flowcytometry on gated lymphocytes.

Example 7 Stability of the Modified hBAT-1

CT-001 mAbs comprising at least one amino acid modification in aposition selected from: Thr 5, Thr 20, Cys 71, Asn 75, Ser 76, Phe 93and Phe 97 of the light chain variable region having the amino acidsequence of SEQ ID NO: 1-4; Asp 54, Ser 55 and Trp 107 of the heavychain variable region having the amino acid sequence of SEQ ID NO: 5-9;Asn 51, Ser 52, Asp 63 and Ser 64 of the light chain constant regionhaving the amino acid sequence of SEQ ID NO: 10; or Thr 3, Ser 7, Asn42, Ser 43, Asn 86, His 87, Asp 104, Lys 105, Thr 108, Met 135, Asp 153,Pro 154, Asp 163, Gly 164, Asn 180, Ser 181, Asn 198, Gly 199, Asn 267,Gly 268, Asp 282, Ser 283, Asp 284, Ser 285, Asn 317, His 318, Lys 330and Met 311 of the heavy chain constant region having the amino acidsequence of SEQ ID NO: 12, are formed as known in the art.

The stability of the modified mAbs is evaluated using variousbiochemical and biophysical techniques that assess size, aggregationstate, structure, and intermolecular contacts of the antibodies as knownin the art. For instance, the purity and aggregation state of theantibodies may be studied by size exclusion chromatography (SEC) andanalytical ultracentrifugation (AUC).

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

What is claimed is:
 1. An antibody or antigen binding fragment thereofcomprising a light chain variable region selected from the groupconsisting of SEQ ID NO: 1, 2, 3, and 4, and a heavy chain variableregion selected from the group consisting of SEQ ID NO: 5, 6, 7, 8, and9, wherein the antibody or antigen binding fragment thereof comprises atleast one amino acid substitution at a position selected from: Phe 97and Phe 93 of the light chain variable region to Ala, Leu or Val, or asubstitution of Trp 107 of the heavy chain variable region to Ala, Leu,Val or Tyr.
 2. The antibody or antigen binding fragment thereof of claim1, comprising an amino acid substitution of Phe 97 of the light chainvariable region and an amino acid substitution of Trp 107 of the heavychain variable region.
 3. The antibody or antigen binding fragmentthereof of claim 1, comprising an amino acid substitution of Phe 93 ofthe light chain variable region and an amino acid substitution of Trp107 of the heavy chain variable region.
 4. The antibody or antigenbinding fragment thereof of claim 1, wherein the light chain variableregion comprises the amino acid sequence selected from the groupconsisting of: SEQ ID NO: 14, 15, 16, 17, 18, 19, 20 and
 21. 5. Theantibody or antigen binding fragment thereof of claim 1, wherein thelight chain variable region comprises the amino acid sequence as setforth in any one of SEQ ID NO: 14 or
 15. 6. The antibody or antigenbinding fragment thereof of claim 1, wherein the light chain variableregion comprises the amino acid sequence as set forth in SEQ ID NO: 29.7. The antibody or antigen binding fragment thereof of claim 1, whereinthe light chain variable region comprises the amino acid sequence as setforth in SEQ ID NO:
 30. 8. The antibody or antigen binding fragmentthereof of claim 1, wherein the heavy chain variable region comprisesthe amino acid sequence selected from the group consisting of: SEQ IDNO: 24, 25, 26, 27 and
 28. 9. The antibody or antigen binding fragmentthereof of claim 1, wherein the heavy chain variable region comprisesthe amino acid sequence as set forth in SEQ ID NO:
 24. 10. The antibodyor antigen binding fragment thereof of claim 1, wherein the heavy chainvariable region comprises the amino acid sequence as set forth in SEQ IDNO:
 31. 11. The antibody or antigen binding fragment thereof of claim 1,further comprising at least one amino acid modification at a positionselected from the group consisting of: Thr 5, Thr 20, Cys 71, Asn 75 andSer 76 of the light chain variable region and Asp 54 and Ser 55 of theheavy chain variable region.
 12. The antibody or antigen bindingfragment thereof of claim 1, comprising a combination of a heavy chainvariable region and a light chain variable region, the combination isselected from the group consisting of: SEQ ID NO: 5/SEQ ID NO: 1, SEQ IDNO: 6/SEQ ID NO: 2, SEQ ID NO: 7/SEQ ID NO: 2, SEQ ID NO: 7/SEQ ID NO:3, SEQ ID NO: 8/SEQ ID NO: 3, and SEQ ID NO: 7/SEQ ID NO:
 1. 13. Theantibody or antigen binding fragment thereof of claim 1 having anantitumor activity of similar, or greater than, mBAT-1.
 14. The antibodyor antigen binding fragment thereof of claim 1 having an antitumoractivity of similar, or greater than, hBAT-1.
 15. The antibody orantigen binding fragment thereof of claim 1, wherein the fragment of thehumanized antibody is selected from the group consisting of: Fv, F(ab'),F(ab')2, and a single chain antibody.
 16. A pharmaceutical compositioncomprising as an active ingredient the antibody of claim 1 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier,diluent or stabilizer.
 17. A method for treating cancer or animmunodeficiency disorder in a subject in need thereof, comprisingadministering to the subject an effective amount of a pharmaceuticalcomposition according to claim
 16. 18. The method of claim 17 whereinthe subject has a non-solid tumor or a hematologic malignancy.
 19. Themethod of claim 17 wherein the subject has a cancer selected from thegroup consisting of a colorectal carcinoma, a non-small lung cancer(NSCLC), a small cell lung cancer (SCLC), a breast carcinoma; amelanoma; an ovarian carcinoma, a cervical carcinoma, a pancreaticcancer, a head and neck carcinoma, a gastrointestinal carcinoma, anesophageal tumor, a hepatocellular carcinoma, multiple myeloma, a renalcell carcinoma, a prostate tumor, non-Hodgkin's lymphoma, Hodgkin'sdisease, mantle cell lymphoma, Kaposi's sarcoma, a squamous cellcarcinoma, a basal cell carcinoma, acute myeloid leukemia (AML), chronicmyelocytic leukemia (CML), acute lymphocytic leukemia (ALL), and chroniclymphocytic leukemia (CLL).
 20. The method of claim 17 wherein thesubject has a cancer selected from the group consisting of: colorectalcarcinoma, melanoma, pancreatic cancer, head and neck carcinoma,esophageal tumor, multiple myeloma, renal cell carcinoma, non-Hodgkin'slymphoma and Hodgkin's disease.